Substrate Transport Research Articles

ATP hydrolytic activity of purified Spf1p correlate with micellar lipid fluidity and is dependent on conserved residues in transmembrane helix M1.

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in pleiotropic phenotypes related to severe ER stress. They were recently proposed to function in peptide translocation although their specificity have yet to be confirmed in reconstituted assays using the purified enzyme. A general theme for P-type ATPases is that binding and transport of substrates is coupled to hydrolysis of ATP in a conserved allosteric mechanism, however several independent reports have shown purified Spf1p to display intrinsic spontaneous ATP hydrolytic activity after purification. It has never been determined to what extend this spontaneous activity is caused by uncoupling of the enzyme. In this work we have purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and have observed that the intrinsic ATP hydrolytic activity of the purified and re-lipidated protein can be stimulated by specific detergents (C12E8, C12E10 and Tween20) in mixed lipid/detergent micelles in the absence of any apparent substrate. We further show that this increase in activity correlate with the reaction temperature and the anisotropic state of the mixed lipid/detergent micelles and further that this correlation relies on three highly conserved phenylalanine residues in M1. This suggests that at least part of the intrinsic ATP hydrolytic activity is allosterically coupled to movements in the TM domain in the purified preparations. It is suggested that free movement of the M1 helix represent an energetic constraint on catalysis and that this constraint likely is lost in the purified preparations resulting in protein with intrinsic spontaneous ATP hydrolytic activity. Removal of the N-terminal part of the protein apparently removes this activity.

Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay—Biological Implications

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.

Diet-induced insulin resistance altered cardiac GLUT4 and FATP/CD36 expression in rats

BackgroundAltered substrate transport protein expression is central to the effect of insulin resistance on cardiac metabolism. The present study was thus designed to investigate the comparative effects of high fat, high sucrose and salt-induced IR on cardiac expression of fatty acid transporter (FATP) and glucose transporter (GLUT4) in rats.ResultsRats fed with high fat, high sucrose and salt diets developed impaired glucose tolerance (p > 0.05) and hyperinsulinemia (p < 0.05) compared with control group. Myocardial glucose transporter expression was significantly increased (p < 0.001 for salt-induced IR; p < 0.01 for sucrose-induced IR; p < 0.01 for fat-induced IR) across all IR groups compared with control. Fatty acid transporter expression was also increased (p < 0.001) in high salt diet-induced IR rats, and high fat diet-induced IR rats (p < 0.05).ConclusionsOur results demonstrate that salt and not caloric excess has a potential role in IR alteration of myocardial substrate transport protein expression in the rat.

Itraconazole-Induced Increases in Gilteritinib Exposure Are Mediated by CYP3A and OATP1B.

Gilteritinib, an FDA-approved tyrosine kinase inhibitor approved for the treatment of relapsed/refractory FLT3-mutated acute myeloid leukemia, is primarily eliminated via CYP3A4-mediated metabolism, a pathway that is sensitive to the co-administration of known CYP3A4 inhibitors, such as itraconazole. However, the precise mechanism by which itraconazole and other CYP3A-modulating drugs affect the absorption and disposition of gilteritinib remains unclear. In the present investigation, we demonstrate that pretreatment with itraconazole is associated with a significant increase in the systemic exposure to gilteritinib in mice, recapitulating the observed clinical drug–drug interaction. However, the plasma levels of gilteritinib were only modestly increased in CYP3A-deficient mice and not further influenced by itraconazole. Ensuing in vitro and in vivo studies revealed that gilteritinib is a transported substrate of OATP1B-type transporters, that gilteritinib exposure is increased in mice with OATP1B2 deficiency, and that the ability of itraconazole to inhibit OATP1B-type transport in vivo is contingent on its metabolism by CYP3A isoforms. These findings provide new insight into the pharmacokinetic properties of gilteritinib and into the molecular mechanisms underlying drug–drug interactions with itraconazole.

Fabrication of a Highly Efficient Wood-Based Solar Interfacial Evaporator with Self-Desalting and Sterilization Performance.

Solar interfacial evaporation based on wood-derived materials has been considered a promising strategy for desalination and wastewater purification. Herein, we adopted delignified wood (DW) as the water transport substrate and lignosulfonate (LS)-modified narrow-band gap semiconductor nickel disulfide (NiS2) as the light-absorbing agent (LS-NiS2) to fabricate a high-efficiency evaporator (LS-NiS2@DW). On the one hand, the high absorbance (>95%) within a broad wavelength range and excellent photothermal conversion efficiency of LS-NiS2 endow efficient solar energy utilization. On the other hand, the hydrophilicity of DW facilitates water activation, which results in a lower evaporation enthalpy of LS-NiS2@DW (1274.4 kJ kg-1) than that of pure water. By combining LS-NiS2 and DW, LS-NiS2@DW achieved an evaporation rate as high as 2.80 kg m-2 h-1 under one sun irradiation (1 kW m-2), and the evaporation efficiency reached 87.4%. Notably, LS-NiS2@DW exhibits a high evaporation rate (2.42-2.69 kg m-2 h-1) in simulated seawater for 24 h with no salt crystals formed on the surface. Moreover, LS-NiS2@DW shows high antibacterial activity with about 90% reduction in bacterial survival rate. This work could provide new perspectives for the design of a high-efficiency wood-based photothermal evaporator.

A method of inhibiting the ABCB1 protein in the blood-brain barrier &lt;i&gt;in vivo&lt;/i&gt;

Introduction. Increased functional activity of the P-glycoprotein transporter (ABCB1) in the blood-brain barrier (BBB) is a possible reason why neuroprotective pharmacotherapy is ineffective after ischaemic stroke.&#x0D; Study aim to develop a way to inhibit the functional activity of ABCB1 at the BBB.&#x0D; Materials and methods. The study was performed on 60 male Wistar rats weighing 200-280 g. The functional activity of ABCB1 at the BBB was assessed by measuring the plasma and cortical levels of the marker transporter substrate fexofenadine (intravenous administration of 10 mg/kg). Thirty minutes before the administration of fexofenadine, 1 ml/kg of intravenous saline (n = 30) or 17.6 mg/kg of omeprazole, the transporter's systemic inhibitor (n = 30), was administered to the rats. The total amount of fexofenadine in the systemic circulation and the cerebral cortex was assessed using high performance liquid chromatography, by calculating the area under the blood concentrationtime curve (AUC0-t(plasma)) or the cerebral cortex concentration (AUC0-t(brain)). BBB permeability was calculated using the ratio AUC0-t(brain)/AUC0-t(plasma).&#x0D; Results. The administration of omeprazole before fexofenadine did not affect the plasma level of the latter at any time point under analysis. Fexofenadines AUC0-t(plasma) also did not differ between the series. However, the administration of omeprazole increased the cortical level of fexofenadine by 2.96 times (p = 0.009), 5 minutes after administration of the latter, and increased the AUC0-t(brain) by 1.49 times (p = 0.012). AUC0-t(brain)/AUC0-t(plasma) increased by 1.71 times when omeprazole was used (p = 0.003). Therefore, omeprazole inhibits the functional activity of ABCB1 at the BBB.&#x0D; Conclusions. We developed and tested a method for inhibiting ABCB1 activity at the BBB.

Determining Substrate Oxygen Transport Resistance at Limiting Current Using Pore Network Modelling

The polymer electrolyte membrane (PEM) fuel is a promising technology to supplant traditional, greenhouse gas emitting energy conversion devices. However, widespread commercial adoption of the PEM fuel cell is hindered by the high cost of the precious metals required for its catalyst (1). The amount of catalyst required in a PEM fuel cell can be reduced if the reactant (oxygen) is delivered more efficiently to the catalyst. Therefore, previous studies have sought to characterize the mechanisms affecting oxygen transport in PEM fuel cells (2)(3)(4)(5). In past studies, the oxygen transport resistance of a PEM fuel cell was measured at limiting current, and this value was further decomposed into contributions from individual PEM fuel components using empirical modelling. By resolving each PEM fuel cell component’s contribution to oxygen transport resistance, an informed approach can be undertaken to design next generation components with enhanced oxygen transport capabilities.In this study, we employed pore network modelling to quantify the oxygen transport resistance arising from the presence of liquid water within the substrate region of the gas diffusion layer (GDL). First, we captured the operando liquid water distribution within a PEM fuel cell operating at limiting current using synchrotron X-ray radiography. Next, we used a combination of invasion percolation and an in-house water invasion inlet selection algorithm to partially saturate the representative pore network of the substrate and to reproduce the mean water saturation. Finally, we obtained substrate oxygen transport resistance values by performing transport simulations on the partially saturated pore network and compare our findings with the experimental observations. References X. X. Wang, M. T. Swihart, and G. Wu, Nature catalysis, 2, 7 (2019).D. R. Baker, D. A. Caulk, K. C. Neyerlin, and M. W. Murphy, J. Electrochem. Soc., 156, B991 (2009).T. Reshtenko, J. St-Pierre, J. Electrochem. Soc., 161, F1089 (2014)D. Muirhead, R. Banerjee, M. G. George, N. Ge, P. Shrestha, H. Liu, J. Lee, A. Bazylak, Electrochimica Acta, 274 (2018).N. Ge, P. Shrestha, M. Balakrishnan, D. Ouellette, A. K. C. Wong, H. Liu, C. H. Lee, J. K. Lee, A. Bazylak, Electrochimica acta, 328 (2019).

Understanding Performance and Durability with KOH and DI-Water Fed Anion Exchange Membrane Electrolyzers

Anion Exchange Membrane electrolyzers (AEMELs) have increased in popularity in recent years due to their potential to combine the benefits from proton exchange membrane electrolyzers and traditional alkaline electrolyzers – namely, high current density operation, pressurized H2 discharge and lowering cost by utilizing low-cost earth abundant electrocatalysts and inexpensive component materials. In the AEMEL, the oxygen evolution reaction (OER) electrode plays a critical role dictating the overall efficiency of the cell due to sluggish OER activity and species transport. In our previous study, we optimized the OER electrode structure by investigating the effects of catalyst loading, catalyst type, the porous transport substrate and additive carbon [1] – resulting in an excellent performance of 1.55 V operation at 1.0 A/cm2.Though that study led to high performance, all of those gains were made using KOH electrolyte being fed to the cell, not deionized (DI) water. The use of DI water complicates AEMEL operation as the liquid phase can no longer be relied on to carry any of the ionic charge – this possibly can reduce the electrochemically active surface area (ECSA) [2]. It has also been stated that pure water operation may have negative consequences on cell durability. Therefore, to allow for AEMELs to operate efficiently on DI water, it is important to understand how the ion transport and charge transfer resistance change as cells are transitioned from KOH to DI water operation. It is also important to understand what effects even trace amounts of KOH can have on behavior. Lastly, the effect of the ionomer properties on the OER anode performance should be well-understood.In this study, we investigate the role of alkaline feed pH on the performance and durability of AEMELs. When shifting from alkaline feed to DI water, it will be shown that there is an increase in ohmic and charge transfer resistance, resulting in significant voltage loss. Because the alkaline feed enhances electrode conductivity by connecting catalyst active sites to the conductive ionomer and AEM [2-3], new electrode structures are needed that allow for enhanced ECSA and lower resistances with DI water feeds. In this work, that was accomplished by creating a four-layer electrode structure as well as manipulating the cell operating conditions. Lastly, our team enabled the use of a high IEC ionomer in the anode catalyst layer by introducing a cross-linker and introducing smaller, cryo-milled ionomer particles. This was important to overcome adhesion issues that can come from high water uptake and swelling in high-IEC ionomers [4-5]. This paper will focus not only on performance, but also longevity, with several cells being stably operated for more than 500 hours continuously.

Functional Inquiry into ATP-Binding Cassette Transporter Genes Contributing to Spirotetramat Resistance in Aphis gossypii Glover.

ATP-binding cassette (ABC) transporters regulate the efflux of a broad spectrum of substrates to extracellular transporting, which play an important role in the detoxification process in arthropods. Here, we described a comprehensive approach to explore the involvement of ABC transporters in spirotetramat resistance in cotton aphids. In this study, synergism bioassays showed 17.05% and 35.42% increases in the toxicity to spirotetramat with the ABC inhibitor verapamil in adult and 3rd instar nymph aphids of the SR strain, respectively. In a competitive assay based on the microinjection of a fluorescent ABC transporter substrate, verapamil (a general ABC inhibitor) and spirotetramat significantly inhibited the elimination of Texas Red. Based on transcriptome data of midguts of spirotetramat-susceptible (SS) and -resistant (SR) strains, the expression levels of ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were significantly upregulated in the SR strain midgut compared to that of the SS strain. Gene functional analysis based on ectopic expression and RNA interference (RNAi) proved that ABCB4, ABCB5, ABCF2, MRP11, and MRP12 were involved in the tolerance of cotton aphids to spirotetramat. Moreover, the upregulated ABCF2, ABCB4, and ABCB5 in the midgut of the SR strain contributed more to the resistance of spirotetramat in in vitro functional analysis. In summary, these results demonstrate that candidate ABC transporter genes in the midgut tissue were involved in spirotetramat resistance, which will help reveal the relationship between ABC transporters and the development of spirotetramat resistance in field populations.

Application of physiologically-based pharmacokinetic model approach to predict pharmacokinetics and drug-drug interaction of rivaroxaban: A case study of rivaroxaban and carbamazepine.

Rivaroxaban (RIV; Xarelto; Janssen Pharmaceuticals, Beerse, Belgium) is one of the direct oral anticoagulants. The drug is a strong substrate of cytochrome P450 (CYP) enzymes and efflux transporters. This study aimed to develop a physiologically-based pharmacokinetic (PBPK) model for RIV. It contained three hepatic metabolizing enzyme reactions (CYP3A4, CYP2J2, and CYP-independent) and two active transporter-mediated transfers (P-gp and BCRP transporters). To illustrate the performance of the developed RIV PBPK model on the prediction of drug-drug interactions (DDIs), carbamazepine (CBZ) was selected as a case study due to the high DDI potential. Our study results showed that CBZ significantly reduces the exposure of RIV. The area under the concentration-time curve from zero to infinity (AUCinf ) of RIV was reduced by 35.2% (from 2221.3 to 1438.7ng*h/ml) and by 25.5% (from 2467.3 to 1838.4ng*h/ml) after the first dose and at the steady-state, respectively, whereas the maximum plasma concentration (Cmax ) of RIV was reduced by 37.7% (from 266.3 to 166.1ng/ml) and 36.4% (from 282.3 to 179.5ng/ml), respectively. The developed PBPK model of RIV could be paired with PBPK models of other interested perpetrators to predict DDI profiles. Further studies investigating the extent of DDI between CBZ and RIV should be conducted in humans to gain a full understanding of their safety and effects.

Naltrexone Transport by a Proton-Coupled Organic Cation Antiporter in hCMEC/D3 Cells, an in Vitro Human Blood-Brain Barrier Model.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

Reshaping the Binding Pocket of the Neurotransmitter:Solute Symporter (NSS) Family Transporter SLC6A14 (ATB0,+) Selectively Reduces Access for Cationic Amino Acids and Derivatives.

SLC6A14 (ATB0,+) is unique among SLC proteins in its ability to transport 18 of the 20 proteinogenic (dipolar and cationic) amino acids and naturally occurring and synthetic analogues (including anti-viral prodrugs and nitric oxide synthase (NOS) inhibitors). SLC6A14 mediates amino acid uptake in multiple cell types where increased expression is associated with pathophysiological conditions including some cancers. Here, we investigated how a key position within the core LeuT-fold structure of SLC6A14 influences substrate specificity. Homology modelling and sequence analysis identified the transmembrane domain 3 residue V128 as equivalent to a position known to influence substrate specificity in distantly related SLC36 and SLC38 amino acid transporters. SLC6A14, with and without V128 mutations, was heterologously expressed and function determined by radiotracer solute uptake and electrophysiological measurement of transporter-associated current. Substituting the amino acid residue occupying the SLC6A14 128 position modified the binding pocket environment and selectively disrupted transport of cationic (but not dipolar) amino acids and related NOS inhibitors. By understanding the molecular basis of amino acid transporter substrate specificity we can improve knowledge of how this multi-functional transporter can be targeted and how the LeuT-fold facilitates such diversity in function among the SLC6 family and other SLC amino acid transporters.

Findings on In Vitro Transporter-Mediated Drug Interactions and Their Follow-Up Actions for Labeling: Analysis of Drugs Approved by US FDA between 2017 and 2021.

Understanding possible follow-up actions on in vitro findings helps determine the necessity of labeling for drug interactions. We analyzed information for in vitro findings on transporter-mediated interactions of drugs approved by the U.S. Food and Drug Administration’s Center for Drug Evaluation and Research for the last five years (i.e., 2017–2021) and their follow-up actions for labeling. Higher R values than the pre-defined cut-off were observed with 3.7–39.1% inhibitor drugs in a simple prediction. Among these drugs, 16–41.7% were labeled with their potential drug interactions, while results of supporting studies or scientific rationales were submitted for the other drugs leading to no interaction labeling. In vitro transporter substrates were reported with 1.7–67.6% of drugs. The interaction labels for these substrate drugs were observed in up to 40% of drugs, while the other drugs were not labeled on the drug interactions with claims for their low interaction potential, evidenced by clinical studies or scientific rationales. The systematic and comprehensive analysis in this study will provide insight into the management of in vitro findings for transporter substrate or inhibitor drugs.

Sodium arsenite but not aluminum chloride stimulates ABC transporter activity in renal proximal tubules of killifish (Fundulus heteroclitus)

ABC export proteins including Multidrug resistance-related protein 2 (Mrp2) serve as detoxification mechanism in renal proximal tubules due to active transport of xenobiotics and metabolic waste products into primary urine. The environmental pollutants aluminum and arsenic interfere with a multitude of regulatory mechanisms in the body and here their impact on ABC transporter function was studied. NaAsO2 but not AlCl3 rapidly stimulated Mrp2-mediated Texas Red (TR) transport in isolated renal proximal tubules from killifish, a well-established laboratory model for the determination of efflux transporter activity by utilizing fluorescent substrates for the ABC transporters of interest and confocal microscopy followed by image analysis. This observed stimulation remained unaffected by the translation inhibitor cycloheximide (CHX), but it was abrogated by antagonists and inhibitors of the endothelin receptor type B (ETB)/nitric oxide synthase (NOS)/protein kinase C (PKC) signaling pathway. NaAsO2-triggered effects were abolished as a consequence of PKCα inhibition through Gö6976 and PKCα inhibitor peptide C2–4. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY 294,002 as well as the mammalian target of rapamycin (mTOR) inhibitor rapamycin suppressed NaAsO2-triggered stimulation of luminal TR transport. In addition, the stimulatory effect of NaAsO2 was abolished by GSK650394, an inhibitor of serum- and glucocorticoid-inducible kinase 1 (SGK1), which is an important downstream target. Environmentally relevant concentrations of NaAsO2 further stimulated transport function of P-glycoprotein (P-gp), Multidrug resistance-related protein 4 (Mrp4) and Breast cancer resistance protein (Bcrp) while AlCl3 was ineffective. To our knowledge, this is the first report engaging in the impact of NaAsO2 on efflux transporter signaling and it may contribute to the understanding of defense mechanisms versus this worrying pollutant.

Chlorpyrifos, permethrin and cyfluthrin effect on cell survival, permeability, and tight junction in an in-vitro model of the human blood-brain barrier (BBB)

The blood-brain barrier (BBB) is a structural and functional interface between the plasma and the human brain. Predictive BBB in-vitro models like immortalized human capillary microvascular endothelial cells (HCMEC/D3) can be used to explore the BBB disruption potential of daily exposed chemicals. The present study was focused on investigating the human BBB permeation potential of one organophosphate pesticide, chlorpyrifos (CPF), and two pyrethroids, permethrin (PMT) and cyfluthrin (CFT). HCMEC/D3 cells were exposed to the chemical and the time-dependent pass across BBB along with permeation coefficient (Papp) was calculated. Transendothelial electrical resistance (TEER) was measured for the cells to check the monolayer formation and later to check the reduction in integrity after chemical exposure. Real time PCR was conducted to investigate the effect of chemicals on the expression BBB´s tight and adherens junction proteins. Calculated Papp value for three chemicals was in the following order: CPF>CFT>PMT, where CPF showed the highest permeation coefficient. TEER calculation showed that the integrity decreased after CPF exposure which was in concordance with Papp value whereas for other chemicals, no change in TEER after exposure was observed. In addition, the transwell study showed a higher efflux ratio (ER) (>2) of CFT indicating that CFT could be a substrate for active transport. For CPF and PMT, ER was less than 2, so no active transport seems to be involved. The evaluation of the mRNA expression analysis revealed a statistically significant decrease in Occludin (OCLN) gene expression for CPF, VE-Cadherin (CDH5) for PMT and Zonula Occludens (ZO1) expression for CFT. Our study showed that CPF has the highest potential for inducing cell death, higher permeation, and capability to induce BBB dysfunction than among the above-mentioned chemicals. Additionally, the results of the permeation study could be useful to build a human PBPK model using in vitro-to-in vivo extrapolation approach.

Polyaniline-Derived Nitrogen-Containing Carbon Nanostructures with Different Morphologies as Anode Modifier in Microbial Fuel Cells.

Anode modification with carbon nanomaterials is an important strategy for the improvement of microbial fuel cell (MFC) performance. The presence of nitrogen in the carbon network, introduced as active nitrogen functional groups, is considered beneficial for anode modification. In this aim, nitrogen-containing carbon nanostructures (NCNs) with different morphologies were obtained via carbonization of polyaniline and were further investigated as anode modifiers in MFCs. The present study investigates the influence of NCN morphology on the changes in the anodic microbial community and MFC performance. Results show that the nanofibrillar morphology of NCNs is beneficial for the improvement of MFC performance, with a maximum power density of 40.4 mW/m2, 1.25 times higher than the anode modified with carbonized polyaniline with granular morphology and 2.15 times higher than MFC using the carbon cloth-anode. The nanofibrillar morphology, due to the well-defined individual nanofibers separated by microgaps and micropores and a better organization of the carbon network, leads to a larger specific surface area and higher conductivity, which can allow more efficient substrate transport and better bacterial colonization with greater relative abundances of Geobacter and Thermoanaerobacter, justifying the improvement of MFC performance.

Conformational transition induced in the aspartate:alanine antiporter by l-Ala binding

An aspartate:alanine antiporter (AspT) from the lactic acid bacterium Tetragenococcus halophilus catalyzes the electrogenic aspartate1-:alanine0 exchange reaction. Our previous kinetic analyses of transport reactions mediated by AspT in reconstituted liposomes suggested that, although the substrate transport reactions are physiologically coupled, the putative binding sites of l-aspartate (-Asp) and l-alanine (-Ala) are independently located on AspT. By using the fluorescent probe Oregon Green maleimide (OGM), which reacts specifically with cysteine, we also found that the presence of l-Asp changes the conformation of AspT. In this study, we conducted an OGM labeling assay in the presence of l-Ala. The labeling efficiency of single cysteine mutants (G62C and P79C) in transmembrane helix 3 of the AspT showed novel patterns depending on the presence of l-Ala or analogs. A concentration-dependent shift of AspT from the conformation in the presence of one substrate to that specific to the substrate added subsequently (l-Ala or l-Asp) was observed. Moreover, size-exclusion-chromatography-based thermostability assays indicated that the thermal stability of AspT in the presence of l-Ala differed from that in the presence of l-Asp. From these results, we concluded that l-Ala binding yields a conformation different from the apo or l-Asp binding conformations.

Comparative genome analyses uncovered the cadmium resistance mechanism of enterobacter cloacae.

Cadmium (Cd) can be transported into plants from polluted soils and may cause animal and human diseases through food chains, which requires the development of highly efficient methods for soil Cd remediation. Although we isolated an Enterobacter cloacae strain Cu6 with Cd resistance, this strain cannot be used for soil Cd remediation due to its lower resistance. Here, we domesticated Cu6 and obtained a highly Cd-resistant strain, LPY6, and found that this strain can attenuate the toxic effects of Cd on wheat seedling growth. We deciphered the high Cd-resistance mechanism of LPY6 by genome comparative and genetic analysis. Compared with Cu6, 75 genes were mutated in LPY6. Thirty-four of these genes were deleted, and 41 had single nucleotide polymorphisms (SNPs). Most of these mutated proteins are involved in basic metabolism, substrate transport, stress response and formate and hydrogen metabolism. RNA quantitative analysis and promoter activity assays showed that the transcription or mRNA levels of two operons (cadA and norVW) in these mutated genes were regulated by Cd, zinc (Zn) or lead (Pb) ions, suggesting that these two operons might be required for Cd, Zn or Pb resistance. Expression of cadA and norVW operons in LPY6 partially recovered Cd susceptibility, demonstrating that CadA and NorVW are involved in Cd resistance in E. cloacae. Our findings illustrate that E. cloacae acquires Cd resistance through different pathways and lay a foundation for developing highly efficient methods for soil Cd remediation.

A dedicated C-6 β-hydroxyacyltransferase required for biosynthesis of the glycolipid anchor for Vi antigen capsule in typhoidal Salmonella

Vi antigen is an extracellular polysaccharide produced by Salmonella enterica Typhi, Citrobacter freundii, and some soil bacteria belonging to the Burkholderiales. In Salmonella Typhi, Vi-antigen capsule protects the bacterium against host defenses, and the glycan is used in a current glycoconjugate vaccine to protect against typhoid. Vi antigen is a glycolipid assembled in the cytoplasm and translocated to the cell surface by an export complex driven by an ABC transporter. In Salmonella Typhi, efficient export and cell-surface retention of the capsule layer depend on a reducing terminal acylated-HexNAc moiety. Although the precise structure and biosynthesis of the acylated terminus has not been resolved, it distinguishes Vi antigen from other known glycolipid substrates for bacterial ABC transporters. The genetic locus for Vi antigen-biosynthesis encodes a single acyltransferase candidate (VexE), which is implicated in the acylation process. Here, we determined the structure of the VexE invitro reaction product by mass spectrometry and NMR spectroscopy to reveal that VexE catalyzes β-hydroxyacyl-ACP dependent acylation of the activated sugar precursor, uridine-5'-diphospho-GlcNAc, at C-6 to form UDP-6-O-[β-hydroxymyristoyl]-α-d-GlcNAc. VexE belongs to the lysophosphatidyl acyltransferase family, and comparison of an Alphafold VexE model to solved lysophosphatidyl acyltransferase structures, together with modeling enzyme:substrate complexes, led us to predict an enzyme mechanism. This study provides new insight into Vi terminal structure, offers a new model substrate to investigate the mechanism of glycolipid ABC transporters, and adds biochemical understanding for a novel reaction used in the synthesis of an important bacterial virulence factor.

Phylogenetic analysis and structural prediction reveal the potential functional diversity between green algae SWEET transporters.

Sugar-Will-Eventually-be-Exported-Transporters (SWEETs) are an important family of sugar transporters that appear to be ubiquitous in all organisms. Recent research has determined the structure of SWEETs in higher plants, identified specific residues required for monosaccharide or disaccharide transport, and begun to understand the specific functions of individual plant SWEET proteins. However, in green algae (Chlorophyta) these transporters are poorly characterised. This study identified SWEET proteins from across representative Chlorophyta with the aim to characterise their phylogenetic relationships and perform protein structure modelling in order to inform functional prediction. The algal genomes analysed encoded between one and six SWEET proteins, which is much less than a typical higher plant. Phylogenetic analysis identified distinct clusters of over 70 SWEET protein sequences, taken from almost 30 algal genomes. These clusters remain separate from representative higher or non-vascular plant SWEETs, but are close to fungi SWEETs. Subcellular localisation predictions and analysis of conserved amino acid residues revealed variation between SWEET proteins of different clusters, suggesting different functionality. These findings also showed conservation of key residues at the substrate-binding site, indicating a similar mechanism of substrate selectivity and transport to previously characterised higher plant monosaccharide-transporting SWEET proteins. Future work is now required to confirm the predicted sugar transport specificity and determine the functional role of these algal SWEET proteins.